Metabolic Glycan Imaging by Isonitriletetrazine Click Chemistry

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DOI: 10.1002/cbic.201300130 Metabolic Glycan Imaging by Isonitrile–Tetrazine Click Chemistry Shaun Stairs,[a] Andr A. Neves,[b] Henning Stçckmann,[a] Yelena A. Wainman,[a] Heather Ireland-Zecchini,[b] Kevin M. Brindle,[b] and Finian J. Leeper*[a]

Bioorthogonal chemistry is the specific and exclusive reaction electrophiles such as maleimides, but the thiol group is also between a probe and a reporter group in the presence of the not truly bioorthogonal and is present on many biomolecules. varied functionality present in biological systems.[1,2] In the An unactivated alkene reacts very slowly with tetrazines,[18] but chemical-reporter strategy, a biologically inert functional group a cyclopropene group reacts considerably faster and has re- is incorporated into the desired biomolecule, and, at the ap- cently been successfully incorporated into cell-surface glycans, propriate time, the reaction with a complementary abiotic initially by using a cyclopropene-conjugated sialic acid[19] but functionality linked to various probes allows specific labelling very recently also with an N-acyl-mannosamine.[20] The azide of the biomolecule.[3] This strategy has been used to label vari- functional group has been most useful for these types of stud- ous biomolecules including proteins, lipids and glycans.[4–7] ies as it is not found in mammals and it undergoes a specific Glycans are complex biological macromolecules consisting 1,3-dipolar cycloaddition with alkynes, either catalysed by of covalently linked scaffolds of monosaccharide units. Most copper or promoted by ring strain. The azide group has also glycans exist as membrane-bound glycoconjugates, but many been used for metabolic labelling of bacterial lipopoly- are secreted and become part of the extracellular matrix. They saccharides.[23] New bioorthogonal chemistries that are com- have important roles in intercellular signalling, cell adhesion patible with the metabolic glycan labelling strategy and are and motility, and have also been shown to play a pivotal role additionally orthogonal to the azide reporter group, are ur- in cancer onset and progression.[8] In many cancer phenotypes, gently required to further facilitate the study of cell-surface certain sugars, particularly sialic acid, are overexpressed, thus glycosylation. glycan imaging has become an attractive target for clinical de- The isonitrile (or isocyano) group is neutral overall and con- velopment.[9,10] We have recently demonstrated that glycan sists of just two atoms. Isonitriles are stable at biologically rele- imaging can be used to detect tumours in vivo.[11] vant pH values and display no appreciable toxicity in mam- Labelling of glycoproteins in living systems presents a partic- mals.[24–26] Like the widely utilised azide group, isonitriles are ular challenge as the chemical reporter must be introduced not found in humans, though they are found in some natural metabolically. This is achieved by feeding to the cells a sugar products from various marine and terrestrial sources.[27,28] Isoni- which is a metabolic precursor with an additional chemical re- triles undergo a [4+1] cycloaddition with tetrazines, which porter group attached. This sugar is then incorporated by the have already been used extensively as bioorthogonal reaction intracellular machinery into the growing glycan. The size and partners for trans-cyclooctenes.[29,30] With many primary and charge of the chemical reporter are of key importance if the secondary isonitriles, a tautomerisation to an imine occurs, and precursor is to be accepted by the host cell’s enzymatic path- in aqueous solution this imine hydrolyses rapidly. However, we ways. Metabolic glycan labelling has currently only been ach- have shown that adducts with tertiary isonitriles, which cannot ieved by using azide,[9,12–14] alkyne,[15] ketone,[16] thiol,[17] tautomerise, are remarkably stable and that, with a primary 3- alkene[18] and cyclopropene[19–21] groups as the chemical report- isocyanopropionyl ester, the imine tautomerises further to er group. The alkyne chemical reporter requires a copper cata- a conjugated enamine, which is slow to hydrolyse.[31] The rates lyst for ligation with an azide, thus its utility for live-cell experi- of reaction of a dipyridyltetrazine probe with primary (3-isocya- ments is limited, although biocompatible copper catalysts are nopropionate) and tertiary isonitriles were 0.12 and being developed.[22] Ketone group labelling is achieved by re- 0.57mÀ1 sÀ1, and the adducts have half-lives of 16 and action with a hydrazide probe; this presents two major prob- 63 hours, respectively, in aqueous solution.[31] Thus, in terms of lems: the reaction is not truly bioorthogonal, as ketones are reactivity and stability of the conjugate, the tertiary isonitrile found in many biomolecules, and reduced pH values are re- would be a more attractive choice for a bioorthogonal ligation, quired for efficient ligation. Thiosugars can be labelled with but its greater size might prevent its metabolic incorporation into glycans by the host cell’s enzymes. [a] S. Stairs, Dr. H. Stçckmann, Y. A. Wainman, Dr. F. J. Leeper We hypothesised that, owing to its small size, stability and Department of Chemistry, University of Cambridge reactivity with tetrazine, the isonitrile group would be an ideal Lensfield Road, Cambridge, CB2 1EW (UK) candidate for a chemical reporter suitable for glycan imaging. E-mail: [email protected] In this paper, we present a proof-of-principle study in which [b] Dr. A. A. Neves, Dr. H. Ireland-Zecchini, Prof. K. M. Brindle isonitrile-bearing sugars are incorporated into cell surfaces and Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE (UK) labelled by a bioorthogonal isonitrile–tetrazine click reaction. Supporting information for this article is available on the WWW under First we checked that both primary and tertiary isonitriles http://dx.doi.org/10.1002/cbic.201300130. are bioorthogonal by incubating them with 10 mm glutathione

2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 0000, 00,1–5 &1& These are not the final page numbers! ÞÞ CHEMBIOCHEM COMMUNICATIONS www.chembiochem.org for 24 h at pH 7.4 and 378C. No reaction was detected by 1H NMR spectroscopy (see the Supporting Information). To begin the study itself, primary and tertiary isonitrile derivatives of glucosamine, galactosamine and mannosamine were synthesised (Scheme 1). Ac4GlcN-n-Iso and Ac4GalN-n-Iso were

Scheme 2. A) Structure of the tetrazine-biotin conjugate. B) Comparison of the shapes of the -NAz and N-n-Iso groups

Lewis lung carcinoma (LL2) cells were seeded at a density of 2104 cells cmÀ2 and incubated for 24 h. The cells were then exposed to either the vehicle (DMSO, <0.25% v/v) or isonitrile sugar at 200 mm and incubated for a further 24 h. The cells were harvested and treated first with 100 mm Tz–biotin for 30 min then, after a series of washes with ice-cold buffer, with neutravidin-DyLight680 for 15 min. After a final set of washes, Scheme 1. Structures of the six isonitrile sugars used in this study. the cells were analysed by flow cytometry (Figure 1). There was no significant difference in the mean fluorescence intensity (MFI) of the tertiary isonitrile sugar pulsed cells and synthesised by 1-ethyl-3-(3-(dimethylamino)propyl)carbodii- the control cells. As it has previously been shown that the click mide hydrochloride (EDC) coupling of the corresponding O- reaction between a similar tetrazine probe and a tertiary isoni- peracetylated, free-amine sugars with 3-formamido- propanoic acid followed by dehydration with phos- phorus oxychloride. Ac4ManN-n-Iso was produced by a similar coupling reaction between mannosamine and 3-(formamido)propanoic acid, followed by pera- cetylation with acetic anhydride and pyridine, and then dehydration of the formamide. The tertiary isonitrile sugars Ac4GlcN-t-Iso, Ac4GalN-t-Iso and

Ac4ManN-t-Iso were formed in an analogous fashion by coupling 2-isocyano-2-methylpropanoic acid with the same amino sugar derivatives (see the Support- ing Information for details). A two-step labelling procedure is normally used to detect incorporation of chemical reporter groups into cell-surface glycans; the cell surface is initially biotinylated by treatment with the chemical reporter. The biotinylated surface can then be visualised by using fluorescently labelled neutravidin followed by confocal microscopy or flow cytometry. This approach improves the signal-to-background ratio (SBR), as it allows one to compensate for the slow rate of the first bioorthogonal reaction by delivering a high concentration of non-fluorogenic probe. The fluorescent component can then be delivered at a much lower concentration, due to the rapid kinet- Figure 1. Flow cytometry results from the two-step labelling procedure. LL2 cells were ics of biotin–neutravidin binding, thus minimising exposed to either vehicle (DMSO, <0.25% v/v, control) or one of the isonitrile sugars. the background signal that arises from nonspecific The mean fluorescence intensity (MFI) of the cells after the labelling procedure is shown. binding. To apply this approach, a tetrazine-biotin Error bars represent standard deviation of three repeats, 10000 events per repeat (see Figures S1 and S2 for details). Numbers above bars are the SBRs (sugar MFI/control MFI) conjugate (Tz–biotin, Scheme 2A) was synthesised and the associated error. *P<0.05, **P<0.005, differences between control and sugar (see the Supporting Information). MFI were significant, two-tailed t-test.

2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 0000, 00,1–5 &2& These are not the final page numbers! ÞÞ CHEMBIOCHEM COMMUNICATIONS www.chembiochem.org trile is bioorthogonal, the sugars had either not been delivered To confirm our findings, quantitative epifluorescence micros- to the cell surface as glycans or the isonitrile group had been copy images of labelled cells were obtained. As little labelling degraded in the process. However, with the primary isonitrile was observed with the tertiary isonitrile sugars, only the pri- sugars, the pulsed cells showed significant SBRs of 7.1(Æ2.0) mary isonitrile sugars were used (Figure 2). As expected, the for Ac4GlcN-n-Iso, 6.9(Æ2.0) for Ac4ManN-n-Iso and 2.1(Æ0.6) for fluorescent labelling was restricted to the cell surfaces. The in-

Ac4GalN-n-Iso. Thus it appears that not only are the 3-isocyano- creases in fluorescence of the pulsed over the unpulsed cells propionyl (n-Iso)-substituted sugars accepted as substrates for (rows A and C) were 2.7-, 5.6- and 5.8-fold for Ac4GalN-n-Iso, the glycan biosynthetic enzymes but also the isonitrile group Ac4GlcN-n-Iso and Ac4ManN-n-Iso, respectively; this closely cor- is sufficiently metabolically stable during the metabolic proc- relates with the values obtained by flow cytometry. essing to be expressed intact on the cell surface. In order to understand why higher concentrations of the ter- A very interesting aspect of these results is the relatively tiary isonitrile (t-Iso) sugars give lower labelling levels, their high incorporation of the glucosamine derivative and low in- effect on cell growth was examined. LL2 cells were grown in corporation of the galactosamine derivative. Bertozzi and co- a 96-well microplate, and the level of confluence was mea- workers have previously shown that it is the other way round sured at three hour intervals over the course of one week (Fig- with azido sugars, and N-azidoacetyl-glucosamine (GlcNAz) is ure S3). The sugars were administered at 200 mm after 24 h poorly incorporated into the cell-surface glycans of human cell and dramatically retarded cell growth and caused a clear lines,[32] whereas GalNAz is well incorporated, and we have had change in morphology (Figure S4). Cells shrank and became the same experience. It has been shown that the action of more circular after 24 h, thus accounting for the slight reduc- UDP-GlcNAc-pyrophosphorylase on GlcNAz 1-phosphate is less tion in confluence. During the flow cytometry experiment efficient than on GlcNAc 1-phosphate, the native substrate.[33,34] (Figure 1), viable cells were selected on the basis of high It has been speculated, however, that UDP-GlcNAz should still NADH autofluorescence and low fluorescence from the cell-im- accumulate due to the lack of feedback inhibition in the hex- permeable nucleic acid stain Sytox Green. With the t-Iso osamine salvage pathway, and so the poor incorporation of sugars, cell viability (64–82 %, Figure S5) was similar to that of GlcNAz into cell-surface proteins might also be due to other the control (86 %), thus suggesting that these sugars are cyto- factors such as inefficient transport, diversion into other meta- static rather than cytotoxic. This cytostatic effect of the tertiary bolic pathways or low tolerance of GlcNAc transferases for isonitrile sugars might be the reason for the lack of sugar in- UDP-GlcNAz.[33] corporation observed in Figure 1. Next, the flow cytometry experiments were repeated with A smaller effect was observed for cells treated with the pri- 100 and 50 mm of the isonitrile sugars (Figure 1). Whereas the mary isonitrile sugars. The cells did grow for about 24 h after incorporation levels of tertiary isonitrile sugars only increased sugar-treatment but more slowly than the control cells, and slightly at a concentration of 100 mm, the levels significantly in- growth ceased after longer periods. The percentage of viable m creased at a concentration of 50 m , with SBRs of 4.0(Æ1.9) for cells was the same for Ac4GlcN-n-Iso- and Ac4ManN-n-Iso-treat-

Ac4GlcN-t-Iso, 3.7(Æ1.8) for Ac4GalN-t-Iso and 3.5(Æ1.7) for ed cells as for control cells (70–77% vs. 84%, Figures S1 and

Ac4ManN-t-Iso. This increase is probably due to the decreased S5). Cells exposed to Ac4GalN-n-Iso were less viable (59%) than toxicity of the tertiary isonitrile sugars at lower concentrations the control cells. This modest toxicity of Ac4GalN-n-Iso could to (see below). Interestingly, the incorporation of primary isoni- some extent explain why incorporation levels were lower than trile sugars at concentrations of 100 or 50 mm was considerably for the corresponding Glc and Man derivatives. Additionally, lower than at 200 mm, with SBRs of 2.5(Æ1.6), 1.5(Æ1.2) and the toxicity experiments were repeated with sugar concentra- m 2.7(Æ1.7) for Ac4GlcN-n-Iso, Ac4GalN-n-Iso and Ac4ManN-n-Iso, tions of 100 and 50 m (Figure S3). The cytostatic effect of the respectively, at 50 mm. tertiary isonitrile sugars was much reduced at lower concentra- m The structural differences between GlcN-n-Iso and GlcNAz tions, particularly in the case of Ac4ManN-t-Iso at 50 m , which, are subtle, as both side chains are of similar length, they are after 24 h, only reduced cells to 70 % of the confluence of both neutral overall and they have the same geometry with the control cells. Large increases in growth were observed at a linearly arranged set of three atoms at the terminus the lower concentrations of Ac4ManN-n-Iso and especially

(Scheme 2B). The first nitrogen atom of the azide group is re- Ac4GalN-n-Iso, but growth was only modestly improved at the placed by a methylene group and consequently the isonitrile is lower concentrations of Ac4GlcN-n-Iso. slightly more bulky, but it would be surprising if this small dif- To test the orthogonality of isonitrile–tetrazine click chemis- ference had a major effect. A more significant difference could try with azide–alkyne click chemistry, a solution of 1-pentyl iso- be the ability of the first nitrogen atom of the azide group to cyanide and tetramethoxydibenzocyclooctyne[35] (TMDIBO; m participate in hydrogen bonding, which is not possible for the each 20 m )inCD3CN/D2O (1:1) was incubated at 37 8C for methylene group of the primary isonitrile. Nevertheless, the re- 24 hours. NMR spectroscopy then showed that no reaction sults show that bioorthogonal glycan labelling by isonitrile–tet- had occurred. The same was found for mixtures of tert-butyl razine click chemistry works well with the primary sugars at isonitrile and TMDIBO and of benzyl azide with both isonitriles high concentrations (200 mm) and, to a lesser extent, with the (see the Supporting Information). As it is known that dibenzo- tertiary sugars at low concentrations (50 mm). This new bioor- cyclooctynes do not react with tetrazines,[36,37] we can say that thogonal ligation could facilitate important advances in the isonitrile–tetrazine click chemistry is orthogonal to azide– understanding of protein glycosylation. alkyne chemistry, provided dibenzocyclooctynes are used.[38]

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Figure 2. Fluorescence imaging and cytometry of LL2 cells metabolically labelled with isonitrile sugars. Cells were incubated with 200 mm of each of the isoni- m trile sugars (Ac4Gal-n-Iso, Ac4Glc-n-Iso or Ac4Man-n-Iso) or with vehicle for 24 h at 378C, then with Tz–biotin (300 m ) or vehicle for 30 min at 378C and then with NA647 (50 mgmLÀl) and DAPI nuclear stain for 15 min at RT. Red: NA647, Blue: DAPI. Scale bars: 100 mm. Images were analysed by quantitative imaging cytometry, and the MFI (bottom) was calculated for each experimental group (A to D) corresponding to the different combinations of the three reagents used. Values plotted in the charts are meanÆSD (3500–5000 events counted per cell culture well). *P<0.0005, differences between groups were significant, two-tailed t-test.

In conclusion, we have shown that isonitrile–tetrazine click While this manuscript was in preparation, two-sugar glycan chemistry can be used to metabolically label cell-surface gly- imaging by using a cyclopropene/tetrazine cycloaddition[20] cans. Ac4GlcN-n-Iso and Ac4ManN-n-Iso show good incorpora- and (even more recently) a linear alkene/tetrazine cycloaddi- [18] tion whereas Ac4GalN-n-Iso only gives a modest twofold in- tion, both combined with azide–alkyne cycloaddition for the crease in fluorescence over untreated cells. We have shown second sugar, have been reported. that the primary isonitrile sugars Ac4GlcN-n-Iso and Ac4ManN- n-Iso are not toxic to cells, even at high concentrations, and Acknowledgements that cells continue to grow for the first 24 hours after treatment (the length of the incubations used in the labelling ex- This work was supported by CRUK (A.A.N., H.S., Y.A.W. and H.I.-Z.) periments). We have also shown that Ac GlcN-t-Iso, Ac GalN-t- 4 4 and EPSRC (S.S.). Iso and Ac4ManN-t-Iso are also incorporated, although only at lower concentrations of sugar, giving SBR’s of around three- fold. This study expands the chemical toolbox for metabolic Keywords: bioorthogonal chemistry · click chemistry · glycan labelling, which until now has been heavily reliant on fluorescent probes · glycans · isonitrile · tetrazines azide–alkyne chemistry. Finally, as isonitrile–tetrazine chemistry is orthogonal to the popular azide–alkyne chemistry, simulta- [1] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113, 2056 – 2075; Angew. Chem. Int. Ed. 2001, 40, 2004 – 2021. neous differential multi-sugar imaging is a real possibility with [2] E. M. Sletten, C. R. Bertozzi, Angew. Chem. 2009, 121, 7108– 7133; this chemistry. Angew. Chem. Int. Ed. 2009, 48, 6974 –6998.

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S. Stairs, A. A. Neves, H. Stçckmann, Seeing the sugar coating: N-Acetyl-glu- Y. A. Wainman, H. Ireland-Zecchini, cosamine and mannosamine derivatives K. M. Brindle, F. J. Leeper* tagged with an isonitrile group are metabolically incorporated into cell-surface && – && glycans and can be detected with a fluo- Metabolic Glycan Imaging by rescent tetrazine. This bioorthogonal Isonitrile–Tetrazine Click Chemistry isonitrile–tetrazine ligation is also orthogonal to the commonly used azide- cyclooctyne ligation, and so will allow simultaneous detection of the incorporation of two different sugars.

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